Hepatocyte growth factor (HGF) is a heparin-binding glycoprotein also known as scatter factor or hepatopoietin-A. HGF that has been first identified as a potent hepatotropic growth factor (Nakamura et al., Nature 342:440 (1989)) is a mesenchymal-derived heparin-binding protein having multiple biological effects such as mitogenesis, motogenesis, and morphogenesis of various types of cells. A gene encoding HGF is located at chromosome 7q21.1, and involves 18 exons and 17 introns (Seki T., et al., Gene 102:213-219 (1991)).
A transcript of about 6 kb is transcribed from the HGF gene, and then a full-length polypeptide HGF precursor (flHGF) composed of 728 amino acids is synthesized therefrom, wherein the flHGF includes the following domains: N-terminal hairpin loop-kringle 1-kringle 2-kringle 3-kringle 4-inactivated serine protease. Simultaneously, several other HGF polypeptide isoforms are synthesized by an alternative splicing of the HGF gene. Known isoforms include deleted variant HGF (deletion of five amino acids from kringle 1 of the full-length HGF), NK1 (N-terminal hairpin loop-kringle 1), NK2 (N-terminal hairpin loop-kringle 1-kringle 2), and NK4 (N-terminal hairpin loop-kringle 1-kringle 2-kringle 3-kringle 4). In addition, there are allelic variants of each isoform. The biologically inactive precursors may be converted into active forms of disulfide-linked heterodimer by protease in serum. In the heterodimers, the alpha chain having a high molecular weight forms four kringle domains and an N-terminal hairpin loop like a pre-activated peptide region of plasminogen. The kringle domains of a triple disulfide-bonded loop structure consisting of about 80 amino acids may play an important role in protein-protein interaction. The low-molecular weight beta chain forms an inactive serine protease-like domain. dHGF consisting of 723 amino acids is a polypeptide with deletion of five amino acids in the first kringle domain of the alpha chain, i.e., F, L, P, S and S, due to alternative splicing between exon 4 and exon 5.
In vivo, two isoforms of HGF (flHGF having 728 amino acids and dHGF having 723 amino acids) are generated through alternative splicing between exon 4 and exon 5. Both of flHGF and dHGF are the same in view of several biological functions, but are different from each other in terms of immunological characteristics and several biological characteristics. For example, flHGF exhibits about 20-fold, 10-fold and 2-fold higher activities than dHGF in terms of promoting DNA synthesis in human umbilical cord venous endothelial cell, arterial smooth muscle cell, and NSF-60 (murine myeloblast cell), respectively. dHGF exhibits about 3-fold and 2-fold higher activities than flHGF in terms of promoting DNA synthesis of LLC-PK1 (pig kidney epithelial cells), and OK (American opossum kidney epithelial cells), and mouse interstitial cells, respectively. In addition, flHGF exhibits 70-fold higher solubility in PBS than dHGF. Several anti-dHGF monoclonal antibodies recognize only dHGF and flHGF or a reduced form of dHGF, which implies that the three-dimensional structures of HGF and dHGF are different.
HGF has been shown to stimulate angiogenesis by regulating the growth of endothelial cells and migration of vascular smooth muscle cells. Due its angiogenic activity, HGF is regarded as one of the promising candidates in therapeutic angiogenesis. “Therapeutic angiogenesis” means an intervention that utilizes angiogenic factors as recombinant proteins or genes, for the treatment of ischemic diseases, such as coronary artery disease (CAD) or peripheral artery disease (PAD). HGF has been also known to stimulate not only the growth but also the migration of endothelial cells (Bussolino et al., J Cell Biol. 119:629 (1992); Nakamura et al., J Hypertens 14:1067 (1996)), and has been tested for its role as a re-endothelialization stimulating agent (Yasuda et al., Circulation 101:2546 (2000); Hayashi et al., Gene Ther 7:1664 (2000)). All of the studies on HGF gene therapy described above have been conducted by using flHGF cDNA encoding 728 amino acids, but not dHGF cDNA encoding 723 amino acids.
Diabetic Neuropathies are serious and dangerous diabetic complications, and, in many cases, they lead to simultaneous occurrence of several types of neuropathies. Diabetic neuropathies are largely classified into polyneuropathy and focal neuropathy. The polyneuropathy includes hyperglycemic neuropathy, distal symmetric polyneuropathy, autonomic neuropathy, acute sensory neuropathy, acute painful sensory neuropathy, chronic sensorimotor neuropathy, and the like. The focal neuropathy includes cranial neuropathy, truncal neuropathy, limb neuropathy, thoracolumbar radiculoneuropathy, lumbosacral radiculoplexus neuropathy, and the like (Andrew J. M. et al., Diabetescare 28:956-962 (2005); J Gareth Llewelyn et al., J Neurol Neurosurg Psychiatry 74:15-19 (2003)). Diabetic Neuropathy has severe pain and loss of mobility as its representative symptoms. According to statistics from the U.S., 60 to 70% of people with diabetes have been known to have diabetic neuropathy (American Diabetes Association (ADA), National Institute of Diabetes and Digestive and Kidney Disease (NIDDK)), and 3.9 million or more diabetic patients aged 40 or over have been known to have diabetic neuropathy. The economic cost of these is estimated to be up to $13.7 billion per year, and this cost is expected to increase continuously.
Currently permitted drugs for diabetic neuropathy are only Lyrica® of Pfizer and Cymbalta® of Eli Lilly. However, these two drugs are merely a kind of painkiller alleviating pains shown in diabetic neuropathy, and may not delay the progress of disease or fundamentally ameliorate symptoms. Besides this medicine treatment, allopathy for pain relief, motor function improvement, and mental stress reduction are being used. There is no fundamental treatment at present, and the control of diabetes through dietary control is the only way to minimize the occurrence of diabetic neuropathy. Therefore, new novel of therapeutic agents capable of suppressing or ameliorating the progress of diabetic neuropathy need to be developed.
Throughout this application, several patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains.